Methods of preparing Tecovirimat

ABSTRACT

Disclosed are methods for the preparation of Tecovirimat for the treatment or prophylaxis of viral infections and diseases associated therewith, particularly those viral infections and associated diseases caused by the orthopoxvirus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/420,728, filed Feb. 10, 2015, now allowed, which was filed as theUnited States national phase of the corresponding internationalapplication number PCT/US2013/054816, filed Aug. 14, 2013, which claimspriority to and benefit of U.S. Provisional Application No. 61/683,905,filed on Aug. 16, 2012, which applications are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

Described herein are methods for the preparation of Tecovirimat for thetreatment or prophylaxis of viral infections and diseases associatedtherewith, particularly those viral infections and associated diseasescaused by the orthopoxvirus. Tecovirimat, with a proprietary name ofST-246®, has a chemical name ofN-[(3aR,4R,4aR,5aS,6S,6aS)-3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)-yl]-4-(trifluoromethyl)-benzamide.

BACKGROUND OF THE INVENTION

The Orthopox genus (Orthopoxviridae) is a member of the Poxviridaefamily and the Choropoxivirinae subfamily. The genus consists ofnumerous viruses that cause significant disease in human and animalpopulations. Viruses in the orthopox genus include cowpox, monkeypox,vaccinia, and variola (smallpox), all of which can infect humans.

The smallpox (variola) virus is of particular importance. Recentconcerns over the use of smallpox virus as a biological weapon haveunderscored the necessity of developing small molecule therapeutics thattarget orthopoxviruses. Variola virus is highly transmissible and causessevere disease in humans resulting in high mortality rates (Henderson etal. (1999) JAMA. 281:2127-2137). Moreover, there is precedent for use ofvariola virus as a biological weapon. During the French and Indian wars(1754-1765), British soldiers distributed blankets used by smallpoxpatients to American Indians in order to establish epidemics (Stern, E.W. and Stern, A. E. 1945. The effect of smallpox on the destiny of theAmerindian. Boston). The resulting outbreaks caused 50% mortality insome Indian tribes (Stern, E. W. and Stern, A. E.). More recently, theSoviet government launched a program to produce highly virulentweaponized forms of variola in aerosolized suspensions (Henderson,supra). Of more concern is the observation that recombinant forms ofpoxvirus have been developed that have the potential of causing diseasein vaccinated animals (Jackson et al. (2001) J. Virol., 75:1205-1210).

The smallpox vaccine program was terminated in 1972; thus, manyindividuals are no longer immune to smallpox infection. Even vaccinatedindividuals may no longer be fully protected, especially against highlyvirulent or recombinant strains of virus (Downie and McCarthy. (1958) JHyg. 56:479-487; Jackson, supra). Therefore, mortality rates would behigh if variola virus were reintroduced into the human population eitherdeliberately or accidentally.

Variola virus is naturally transmitted via aerosolized droplets to therespiratory mucosa where replication in lymph tissue producesasymptomatic infection that lasts 1-3 days. Virus is disseminatedthrough the lymph to the skin where replication in the small dermalblood vessels and subsequent infection and lysis of adjacent epidermalcells produces skin lesions (Moss, B. (1990) Poxviridae and TheirReplication, 2079-2111. In B. N. Fields and D. M. Knipe (eds.), FieldsVirology. Raven Press, Ltd., New York). Two forms of disease areassociated with variola virus infection; variola major, the most commonform of disease, which produces a 30% mortality rate and variola minor,which is less prevalent and rarely leads to death (<1%). Mortality isthe result of disseminated intravascular coagulation, hypotension, andcardiovascular collapse, that can be exacerbated by clotting defects inthe rare hemorrhagic type of smallpox (Moss, supra).

A recent outbreak of monkeypox virus underscores the need for developingsmall molecule therapeutics that target viruses in the orthopox genus.Appearance of monkeypox in the US represents an emerging infection.Monkeypox and smallpox cause similar diseases in humans, howevermortality for monkeypox is lower (1%).

Vaccination is the current means for preventing orthopox virus disease,particularly smallpox disease. The smallpox vaccine was developed usingattenuated strains of vaccinia virus that replicate locally and provideprotective immunity against variola virus in greater than 95% ofvaccinated individuals (Modlin (2001) MMWR (Morb Mort Wkly Rep)50:1-25). Adverse advents associated with vaccination occur frequently(1:5000) and include generalized vaccinia and inadvertent transfer ofvaccinia from the vaccination site. More serious complications such asencephalitis occur at a rate of 1:300,000, which are often fatal(Modlin, supra). The risk of adverse events is even more pronounced inimmunocompromised individuals (Engler et al. (2002) J Allergy ClinImmunol. 110:357-365). Thus, vaccination is contraindicated for peoplewith AIDS or allergic skin diseases (Engler et al.). While protectiveimmunity lasts for many years, the antibody response to smallpoxvaccination is significantly reduced 10 to 15 years post inoculation(Downie, supra). In addition, vaccination may not be protective againstrecombinant forms of orthopoxvirus. A recent study showed thatrecombinant forms of mousepox virus that express IL-4 cause death invaccinated mice (Jackson, supra). Given the side effects associated withvaccination, contraindication of immunocompromised individuals, andinability to protect against recombinant strains of virus, betterpreventatives and/or new therapeutics for treatment of smallpox virusinfection are needed.

Vaccinia virus immunoglobulin (VIG) has been used for the treatment ofpost-vaccination complications. VIG is an isotonic sterile solution ofimmunoglobulin fraction of plasma derived from individuals who receivedthe vaccinia virus vaccine. It is used to treat eczema vaccinatum andsome forms of progressive vaccinia. Since this product is available inlimited quantities and difficult to obtain, it has not been indicatedfor use in the event of a generalized smallpox outbreak (Modlin, supra).

Cidofovir ([(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine][HBMPC]) is a nucleoside analog approved for treatment of CMV retinitisin AIDS patients. Cidofovir has been shown to have activity in vitroagainst a number of DNA containing viruses including adenovirus,herpesviruses, hepadnaviruses, polyomaviruses, papillomaviruses, andorthopoxviruses (Bronson et al. (1990) Adv. Exp. Med. Biol. 278:277-83;De Clercq et al. (1987) Antiviral Res. 8:261-272; de Oliveira et al.(1996) Antiviral Res. 31:165-172; Snoeck et al. (2001) Clin Infect. Dis.33:597-602). Cidofovir has also been found to inhibit authentic variolavirus replication (Smee et al. (2002) Antimicrob. Agents Chemother.46:1329-1335).

However, cidofovir administration is associated with a number of issues.Cidofovir is poorly bioavailable and must be administered intravenously(Laezari et al. (1997) Ann. Intern. Med. 126:257-263). Moreover,cidofovir produces dose-limiting nephrotoxicity upon intravenousadministration (Lalezari et al.). In addition, cidofovir-resistance hasbeen noted for multiple viruses. Cidofovir-resistant cowpox, monkeypox,vaccinia, and camelpox virus variants have been isolated in thelaboratory by repeated passage in the presence of drug (Smee, supra).Cidofovir-resistance represents a significant limitation for use of thiscompound to treat orthopoxvirus replication. Thus, the poorbioavailability, need for intravenous administration, and prevalence ofresistant virus underscores the need for development of additional andalternative therapies to treat orthopoxvirus infection.

In addition to viral polymerase inhibitors such as cidofovir, a numberof other compounds have been reported to inhibit orthopoxvirusreplication (De Clercq. (2001) Clin Microbiol. Rev. 14:382-397).Historically, methisazone, the prototypical thiosemicarbazone, has beenused in the prophylactic treatment of smallpox infections (Bauer et al.(1969) Am. J Epidemiol. 90:130-145). However, this compound class hasnot garnered much attention since the eradication of smallpox due togenerally unacceptable side effects such as severe nausea and vomiting.Mechanism of action studies suggest that methisazone interferes withtranslation of L genes (De Clercq (2001), supra). Like cidofovir,methisazone is a relatively non-specific antiviral compound and caninhibit a number of other viruses including adenoviruses,picornaviruses, reoviruses, arboviruses, and myxoviruses (Id.).

Another class of compounds potentially useful for the treatment ofpoxviruses is represented by inhibitors of S-adenosylhomocysteinehydrolase (SAH). This enzyme is responsible for the conversion ofS-adenosylhomocysteine to adenosine and homocysteine, a necessary stepin the methylation and maturation of viral mRNA. Inhibitors of thisenzyme have shown efficacy at inhibiting vaccinia virus in vitro and invivo (De Clercq et al. (1998) Nucleosides Nucleotides. 17:625-634.).Structurally, all active inhibitors reported to date are analogues ofthe nucleoside adenosine. Many are carbocyclic derivatives, exemplifiedby Neplanacin A and 3-Deazaneplanacin A. While these compounds haveshown some efficacy in animal models, like many nucleoside analogues,they suffer from general toxicity and/or poor pharmacokinetic properties(Coulombe et al. (1995) Eur. J Drug Metab Pharmacokinet. 20:197-202;Obara et al. (1996) J Med. Chem. 39:3847-3852). It is unlikely thatthese compounds can be administered orally, and it is currently unclearwhether they can act prophylactically against smallpox infections.Identification of non-nucleoside inhibitors of SAH hydrolase, and otherchemically tractable variola virus genome targets that are orallybioavailable and possess desirable pharmacokinetic (PK) and absorption,distribution, metabolism, excretion (ADME) properties would be asignificant improvement over the reported nucleoside analogues. Insummary, currently available compounds that inhibit smallpox virusreplication are generally non-specific and suffer from use limitingtoxicities and/or questionable efficacies.

In U.S. Pat. No. 6,433,016 (Aug. 13, 2002) and U.S. ApplicationPublication 2002/0193443 A1 (published Dec. 19, 2002) a series ofimidodisulfamide derivatives are described as being useful fororthopoxvirus infections.

New therapies and preventatives are clearly needed for infections anddiseases caused by orthopoxvirus infection.

The co-owned PCT application WO 2004/112718 (published Dec. 29, 2004)discloses the use of di, tri, and tetracyclic acylhydrazide derivativesand analogs, as well as pharmaceutical compositions containing the same,for the treatment or prophylaxis of viral infections and diseasesassociated therewith, particularly those viral infections and associateddiseases caused by the orthopoxvirus. The co-owned U.S. Patentapplication 2008/0004452 (published Jan. 3, 2008) further discloses aprocess for producing ST-246. However, the current process encountersdiastereoselectivity (endo vs. exo), low yields for some steps, use of agenotoxic compound and a very hydroscopic anhydride, and difficulties insecuring some raw materials. Thus, there remains an urgent need todevelop more effective processes for producing ST-246

SUMMARY OF THE INVENTION

The present invention provides a process for making ST-246 outlined inScheme 1

The present invention also provides a process for making ST-246 outlinedin Scheme 2

The present invention further provides a process for making ST-246outlined in Scheme 3

The present invention also provides a process for making ST-246 outlinedin Scheme 4

The present invention further provides a process for making ST-246outlined in Scheme 5

The present invention also provides the following compounds useful inthe synthesis of ST-246:

(a) Compound 6 having the following formula:

(b) Compound 9 having the following formula:

(c) Compound 10 having the following formula:

and

(d) Compound 13 having the following formula:

DETAILED DESCRIPTION OF THE INVENTION

Described herein are processes for producing ST-246. The chemical namefor ST-246 isN-[(3aR,4R,4aR,5aS,6S,6aS)-3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)-yl]-4-(trifluoromethyl)-benzamideand has the following formula:

Accordingly, it has been discovered that ST-246 can be prepared by aprocess called Synthetic Route I, said process comprising:

(a) reacting compound 3 of formula:

with tert-butyl carbazate (compound 5) to form compound 6 of formula:

(b) reacting compound 6 with an acid to form compound 7 or salt thereofof formula:

(c) reacting compound 7 with 4-(trifluoromethyl)benzoyl chloride(compound 8); and

(d) collectingN-[(3aR,4R,4aR,5aS,6S,6aS)-3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)-yl]-4-(trifluoromethyl)-benzamide.

For Synthetic Route I, the acid in step (b) is preferably HCl. Alsopreferably, compound 6 is dissolved in in i-PrOAc prior to the reactionof step (b). Again preferably, a base is present in the reaction of step(c), wherein said base is selected from the group consisting of:pyridine, 4-dimethylaminopyridine, triethylamine anddiisopropylethylamine. Step (c) is preferably carried out at atemperature of less than about 20° C.

It has been also discovered that ST-246 can be prepared by a processcalled Synthetic Route II, said process comprising:

(a) reacting compound 4 of formula:

with maleic anhydride (compound 2) to form compound 9 of formula:

(b) reacting compound 9 with cycloheptatriene (compound 1); and

(c) collectingN-[(3aR,4R,4aR,5aS,6S,6aS)-3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)-yl]-4-(trifluoromethyl)-benzamide.

For Synthetic Route II, step (a) is preferably carried out in o-xyleneand reactants heated to reflux. Also preferably, step (b) is carried outin toluene at a temperature of at least about 75° C.

It has been further discovered that ST-246 can be prepared by a processcalled Synthetic Route III, said process comprising:

(a) reacting maleic anhydride (compound 2) and tert-butyl carbazate(compound 5) to form compound 10 of formula:

(b) reacting compound 10 with an acid to form compound 11 or saltthereof of formula:

(c) reacting compound 11 with 4-(trifluoromethyl)benzoyl halide(compound 8) to form compound 9 of formula:

(d) reacting compound 9 with cycloheptatriene (compound 1); and

(e) collectingN-[(3aR,4R,4aR,5aS,6S,6aS)-3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)-yl]-4-(trifluoromethyl)-benzamide.

For Synthetic Route III, step (a) is preferably carried out in anhydroustoluene under nitrogen atmosphere and reactants heated to reflux. Alsopreferably, the acid in step (b) is HCl. It is also preferred thatcompound 10 is dissolved in i-PrOAc prior to the reaction of step (b).Furthermore, a base is preferably present in the reaction of step (c),wherein said base is selected from the group consisting of: pyridine,4-dimethylaminopyridine, triethylamine and diisopropylethylamine. Alsopreferably, the 4-(trifluoromethyl)benzoyl halide is4-(trifluoromethyl)benzoyl chloride. Step (c) is preferably carried outat a temperature of about 10 to about 25° C. and step (d) is carried outin toluene under nitrogen atmosphere at a temperature above about 110°C.

It has been further discovered that ST-246 can be prepared by a processcalled Synthetic Route IV, said process comprising:

(a) reacting maleic anhydride (compound 2) and tert-butyl carbazate(compound 5) to form compound 10 of formula:

(b) reacting compound 10 with cycloheptatriene (compound 1) to formcompound 6 with the formula:

(c) reacting compound 6 with an acid to form compound 7 or salt thereofof formula:

(d) reacting compound 7 with 4-(trifluoromethyl)benzoyl chloride(compound 8); and

(e) collectingN-[(3aR,4R,4aR,5aS,6S,6aS)-3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)yl]-4-(trifluoromethyl)-benzamide.

For Synthetic Route IV, step (a) is preferably carried out in anhydroustoluene under nitrogen atmosphere and reactants heated to reflux. Alsopreferably, step (b) is carried out under nitrogen atmosphere at atemperature of at least about 75° C. The acid in step (c) is preferablyHCl. It is also preferred that compound 6 is dissolved in in i-PrOAcprior to the reaction of step (c). Also preferably, a base is present inthe reaction of step (d), wherein said base is selected from the groupconsisting of: pyridine, 4-dimethylaminopyridine, triethylamine anddiisopropylethylamine. Step (d) is carried out at a preferredtemperature of less than about 20° C.

It has been further discovered that ST-246 can be prepared by a processcalled Synthetic Route V, said process comprising:

(a) reacting compound 7 having formula:

with 4-Iodobenzoyl chloride (compound 12) to form compound 13 havingformula:

(b) reacting compound 13 with methyl 2,2-difluoro-2-(fluorosulfonyl)acetate; and

(c) collectingN-[(3aR,4R,4aR,5aS,6S,6aS)-3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)-yl]-4-(trifluoromethyl)-benzamide.

For synthetic Route V, a base is preferably present in the reaction ofstep (a), wherein said base is selected from the group consisting of:pyridine, 4-dimethylaminopyridine, triethylamine anddiisopropylethylamine. Also preferably, step (a) is carried out undernitrogen atmosphere at a temperature below about 20° C. and step (b) iscarried out in the presence of dimethylformamide, methyl 2,2-difluoro-2-(fluorosulfonyl)acetate and copper (I) iodide.

Optionally, the ST-246 collected in each of the Synthetic Routes I-Vstep is further purified by column chromatography.

Example 1: Synthetic Route I

Step A. Synthesis of Compound 6 (P=Boc)

To a mixture of compound 3 (5.0 g, 26.3 mmol, synthesized according toWO04112718) in EtOH (80 mL, EMD, AX0441-3) was added tert-butylcarbazate 5 (3.65 g, 27.6 mmol, Aldrich, 98%). The reaction mixture washeated to reflux for 4 h under nitrogen atmosphere. LC-MS analysis ofthe reaction mixture showed less than 5% of compound 3 remained. Thereaction mixture was evaporated under reduced pressure. The residue wasrecrystallized from EtOAc—hexanes, the solid was filtered, washed withhexanes (50 mL) and dried under vacuum to afford compound 6 (3.1 g, 39%yield) as a white solid. The filtrate was concentrated and purified bycolumn chromatography eluting with 25% EtOAc in hexanes to give anadditional 3.64 g (46% yield) of compound 6 as a white solid. Totalyield: 6.74 g (84% yield). ¹H NMR in CDCl₃: δ 6.30 (br s, 1H), 5.79 (t,2H), 3.43 (s, 2H), 3.04 (s, 2H), 1.46 (s, 9H), 1.06-1.16 (m, 2H),0.18-0.36 (m, 2H); Mass Spec: 327.2 (M+Na)⁺

Step B. Synthesis of Compound 7 (HCl Salt)

Compound 6 (3.6 g, 11.83 mmol) was dissolved in i-PrOAc (65 mL, Aldrich,99.6%). 4M HCl in dioxane (10.4 mL, 41.4 mmol, Aldrich) was addeddrop-wise to the above solution keeping the temperature below 20° C. Thereaction mixture was stirred at room temperature overnight (18 h) undernitrogen atmosphere. The resulting solid was filtered, washed withi-PrOAc (15 mL) and dried under vacuum to yield HCl salt of compound 7(1.9 g, 67% yield) as a white solid. The filtrate was concentrated to ⅓its volume and stirred at 10-15° C. for 30 min. The solid was filtered,washed with minimal volume of i-PrOAc and dried to afford additional 0.6g (21% yield) of compound 7. Total yield: 2.5 g (88% yield). ¹H NMR inDMSO-d6: δ 6.72 (br s, 3H), 5.68 (m, 2H), 3.20 (s, 2H), 3.01 (s, 2H),1.07-1.17 (m, 2H), 0.18-0.29 (m, 1H), −0.01-0.07 (m, 1H); Mass Spec:205.1 (M+H)⁺

Step C. Synthesis of ST-246

To a mixture of compound 7 (0.96 g, 4 mmol) in dry dichloromethane (19mL) was added triethylamine (1.17 mL, 8.4 mmol, Aldrich) keeping thetemperature below 20° C. The resulting solution was stirred for 5minutes at 15-20° C., to it was added drop-wise4-(trifluoromethyl)benzoyl chloride 8 (0.63 mL, 4.2 mmol, Aldrich, 97%)and the reaction mixture was stirred at room temperature overnight (18h). LC-MS and TLC analysis showed the correct molecular weight and R_(f)value of ST-246 but the reaction was not complete. Additional 0.3 mL (2mmol, 0.5 eq) of 4-(trifluoromethyl)benzoyl chloride 8 was added to thereaction mixture at 15-20° C. The reaction was then stirred at roomtemperature overnight (19 h). LC-MS analysis indicated ca. 5% ofstarting material 7 still remained. The reaction was stopped anddichloromethane (30 mL) was added. The organic phase was washed withwater (30 mL), saturated aqueous NH₄Cl (30 mL), water (15 mL) andsaturated aqueous NaHCO₃ (30 mL). The organic phase was separated, driedover Na₂SO₄, filtered and concentrated to give crude product. The crudeproduct was purified by column chromatography eluting with 30-50% EtOAcin hexanes to afford ST-246 (0.34 g, 23% yield) as an off-white solid.Analytical data (¹H NMR, LC-MS and HPLC by co-injection) were matchedwith those of ST-246 synthesized according to WO04112718 and wereconsistent.

Example 2: Synthetic Route II

Step A. Synthesis of Compound 9

A mixture of compound 4 (2.0 g, 9.8 mmol) and maleic anhydride 2 (0.96g, 9.8 mmol, Aldrich powder, 95%) in o-xylene (100 mL, Aldrichanhydrous, 97%) was heated to reflux using a Dean-Stark trap apparatusovernight. After 18 h, LC-MS analysis at 215 nm showed the desiredproduct 9 (86%), an uncyclized product (2.6%) and a dimer by-product(11.6%).

The reaction mixture was cooled to 45° C. and evaporated under reducedpressure. The residue was dissolved in EtOAc (50 mL) and the insolublesolid (mostly uncyclized product) was removed by filtration. Thefiltrate was concentrated and purified by column chromatography elutingwith 50% EtOAc in hexanes to yield compound 9 (1.5 g, 54% yield) as anoff-white solid. ¹H NMR in CDCl₃: δ 8.44 (s, 1H), 7.91 (d, 2H), 7.68 (d,2H), 6.88 (s, 2H); Mass Spec: 285.1 (M+H)⁺

Step B. Synthesis of ST-246 (Route II)

A mixture of compound 9 (0.97 g, 3.4 mmol) and cycloheptatriene 1 (0.51mL, 4.42 mmol, distilled before use, Aldrich tech 90%) in toluene (50mL, Aldrich anhydrous) was heated at 95° C. under nitrogen atmosphere.After 1.5 h at 95° C., LC-MS analysis at 254 nm showed 29% conversion tothe desired product (endo:exo=94:6). The resulting solution wascontinued to be heated at same temperature overnight. After 18 h at 95°C., LC-MS analysis indicated 75% conversion with an endo:exo ratio of94:6. The reaction temperature was increased to 110° C. and the reactionwas monitored. After heating at 110° C. for 7 h, LC-MS analysis at 254nm showed 96.4% conversion to the desired product (endo:exo=94:6). Thevolatiles were removed by evaporation under reduced pressure and thereside was purified by column chromatography eluting with 30% EtOAc inhexanes to afford ST-246 (0.29 g, 22.6% yield, HPLC area 99.7% pure and100% endo isomer) as a white solid. Analytical data (¹H NMR, LC-MS andHPLC by co-injection) were matched with those of ST-246 synthesizedaccording to WO04112718 and were consistent. An additional 0.5 g ofST-246 (38.9% yield, endo:exo=97:3) was recovered from columnchromatography. Total Yield: 0.84 g (65.4% yield). ¹H NMR of ST-246 exoisomer in CDCl₃: δ 8.62 (s, 1H), 7.92 (d, 2H), 7.68 (d, 2H), 5.96 (m,2H), 3.43 (s, 2H), 2.88 (s, 2H), 1.17 (s, 2H), 0.24 (q, 1H), 0.13 (m,1H); Mass Spec: 377.1 (M+H)⁺

Example 3: Synthetic Route III

Step A. Synthesis of Compound 10

A mixture of maleic anhydride 2 (15.2 g, 155 mmol, Aldrich powder 95%)and tert-butyl carbazate 5 (20.5 g, 155 mmol, Aldrich, 98%) in anhydroustoluene (150 mL, Aldrich anhydrous) was heated to reflux using aDean-Stark trap apparatus under nitrogen atmosphere. After refluxing for2 h, no starting material 2 remained and LC-MS analysis at 254 nm showedthe desired product 10 (20% by HPLC area), imine by-product (18%) anddisubstituted by-product (56%). The reaction mixture was concentratedand purified by column chromatography eluting with 25% EtOAc in hexanesto afford compound 10 (5.98 g, 18% yield, HPLC area >99.5% pure) as awhite solid. ¹H NMR in DMSO-d6: δ 9.61 (s, 1H), 7.16 (s, 2H), 1.42 (s,9H); Mass Spec: 235.1 (M+Na)⁺.

Step B. Synthesis of Compound 11 (HCl Salt)

Compound 10 (3.82 g, 18 mmol) was dissolved in i-PrOAc (57 mL, Aldrich,99.6%). 4M HCl in dioxane (15.8 mL, 63 mmol, Aldrich) was addeddrop-wise to the above solution keeping the temperature below 20° C. Thesolution was stirred overnight (24 h) at room temperature under nitrogenatmosphere. The resulting solid was filtered, washed with i-PrOAc (10mL) and dried at 45° C. under vacuum for 1 h to afford HCl salt ofcompound 11 (2.39 g, 89% yield) as a white solid. ¹H NMR in CD₃OD: δ6.98 (s, 2H); Mass Spec: 113.0 (M+H)⁺

Step C. Synthesis of Compound 9 (Route III)

To a mixture of compound 11 (1.19 g, 8 mmol) in dry dichloromethane (24mL) was added diisopropylethylamine (2.93 mL, 16.8 mmol, Aldrichredistilled grade) keeping the temperature below 20° C. The resultingsolution was stirred for 5 minute at 15-20° C. and to it was added4-(trifluoromethyl)benzoyl chloride 8 (1.31 mL, 8.8 mmol, Aldrich, 97%)drop-wise. The reaction was stirred at room temperature for 5 h. LC-MSanalysis showed the correct MW but the reaction was not complete.Additional 0.48 mL (0.4 equiv) of 4-(trifluoromethyl)benzoyl chloride 8was added to the reaction mixture at 15-20° C. and the reaction mixturewas stirred at room temperature overnight (21 h). The reaction wasstopped and dichloromethane (50 mL) was added. The organic phase waswashed with water (50 mL), saturated aqueous NH₄Cl (50 mL), water (30mL) and saturated aqueous NaHCO₃ (30 mL). The organic phase wasseparated, dried over Na₂SO₄, filtered and concentrated to give crudeproduct. The crude product was purified by column chromatography elutingwith 30-35% EtOAc in hexanes to afford compound 9 (0.8 g, 35% yield) asa light pink solid. Analytical data (¹H NMR and LC-MS) were consistentwith those of compound 9 obtained in Synthetic Route II.

Step D. Synthesis of ST-246 (Route III)

A mixture of compound 9 (0.5 g, 1.76 mmol) and cycloheptatriene 1 (0.33mL, 3.17 mmol, distilled before to use, Aldrich tech 90%) in toluene (10mL, Aldrich anhydrous) was heated at 110-115° C. under nitrogenatmosphere. After 6 h, LC-MS analysis at 254 nm showed 95% conversion tothe desired product (endo:exo=94:6). The resulting solution was heatedat same temperature overnight (22 h). LC-MS analysis at 254 nm showed nostarting material 9 remained and the desired product (endo:exo=93:7).The reaction mixture was concentrated and purified by columnchromatography eluting with 25-35% EtOAc in hexanes to afford ST-246(0.39 g, HPLC area >99.5% pure with a ratio of endo:exo=99:1) as a whitesolid. Analytical data (¹H NMR, LC-MS and HPLC by co-injection) werecompared with those of ST-246 synthesized according to WO04112718 andwere found to be consistent. An additional 0.18 g of ST-246 (HPLCarea >99.5% pure, endo:exo=91:9) was recovered from columnchromatography. Total Yield: 0.57 g (86% yield).

Example 4; Synthetic Route IV

Step A. Synthesis of Compound 10

A mixture of maleic anhydride 2 (3.4 g, 34.67 mmol, Aldrich powder, 95%)and tert-butyl carbazate 5 (4.6 g, 34.67 mmol, Aldrich, 98%) inanhydrous toluene (51 mL, Aldrich) was heated to reflux using aDean-Stark trap apparatus under nitrogen atmosphere. After refluxing for2.5 h, no starting material 2 remained and LC-MS analysis at 254 nmshowed the desired product 10 (19% HPLC area), imine by-product (18%)and another by-product (56%). The reaction mixture was concentrated andpurified by column chromatography eluting with 30% EtOAc in hexanes toafford compound 10 (1.0 g, 13.6% yield, HPLC area >99% pure) as a whitesolid. Analytical data (¹H NMR and LC-MS) were consistent with those ofcompound 10 obtained in Synthetic Route III.

Step B. Synthesis of Compound 6

A mixture of compound 10 (4.4 g, 20.74 mmol) and cycloheptatriene 1(3.22 mL, 31.1 mmol, distilled before to use, Aldrich tech 90%) intoluene (88 mL, 20 volume, Aldrich anhydrous) was heated at 95° C. undernitrogen atmosphere. After 15 h at 95° C., LC-MS analysis showed 83%conversion to the desired product. The reaction mixture was heated at105° C. overnight. After total 40 h at 95-105° C., LC-MS analysis at 254nm showed ˜99% conversion to the desired product (endo:exo=93:7). Thereaction mixture was concentrated and the crude was purified by columnchromatography eluting with 25-50% EtOAc in hexanes to afford compound 6(2.06 g, 32.6% yield, HPLC area 99.9% pure and 100% endo isomer) as awhite solid. ¹H NMR and LC-MS were consistent with those of compound 6obtained in Synthetic Route I. An additional 4.0 g of 6 (63.4% yield,HPLC area 93% pure with a ratio of endo:exo=91:9) was recovered fromcolumn chromatography. Total Yield: 6.06 g (96% yield).

Step C. Synthesis of Compound 7 (HCl Salt)

Compound 6 (2.05 g, 6.74 mmol) was dissolved in i-PrOAc (26 mL, Aldrich,99.6%). 4M HCl in dioxane (5.9 mL, 23.58 mmol, Aldrich) was addeddrop-wise to the above solution keeping the temperature below 20° C. Thesolution was stirred overnight (18 h) at room temperature under nitrogenatmosphere. The resulting solid was filtered, washed with i-PrOAc (5 mL)and dried under vacuum to yield HCl salt of compound 7 (1.57 g, 97%yield) as a white solid. Analytical data (¹H NMR and LC-MS) wereconsistent with those of compound 7 in Synthetic Route I.

Step D. Synthesis of ST-246 (Route IV)

To a mixture of compound 7 (0.84 g, 3.5 mmol) in dichloromethane (13 mL)was added diisopropylethylamine (1.34 mL, 7.7 mmol) keeping thetemperature below 20° C. and the resulting solution was stirred for 5-10minutes. 4-(Trifluoromethyl)benzoyl chloride 8 (0.57 mL, 3.85 mmol,Aldrich, 97%) was added to above solution keeping the temperature below20° C. The reaction mixture was stirred at room temperature for 2 h.Additional 0.2 mL (0.4 equiv) of 4-(trifluoromethyl)benzoyl chloride 8was added to the reaction keeping the temperature below 20° C. Thereaction was stirred at room temperature overnight (24 h). The reactionmixture was diluted with dichloromethane (20 mL). The organic phase waswashed with water (20 mL), saturated aqueous NH₄Cl (20 mL), water (20mL) and saturated aqueous NaHCO₃ (20 mL). The organic phase wasseparated, dried over Na₂SO₄, filtered and concentrated to give crudeproduct. The crude product was purified by column chromatography elutingwith 30-35% EtOAc in hexanes to afford ST-246 (0.25 g, 19% yield, HPLCarea >99.5% pure) as a white solid. Analytical data (¹H NMR and LC-MS)were consistent with those of ST-246 synthesized according toWO04112718.

Example 5: Synthetic Route V

Step A. Synthesis of Compound 13

To a mixture of compound 7 (1.6 g, 6.65 mmol, synthesized according toSynthetic Route I) in dichloromethane (80 mL,) was added triethylamine(2.04 mL, 14.63 mmol) keeping the temperature below 20° C. and theresulting solution was stirred for 5-10 minute. 4-Iodobenzoyl chloride12 (1.95 g, 7.31 mmol, 1.1 equiv, Aldrich) was added portion-wise undernitrogen atmosphere to the above solution keeping the temperature below20° C. The reaction mixture was stirred at room temperature overnight.After 17 h and 19 h, additional 0.35 g (0.2 equiv) of acid chloride 12was added to the reaction keeping the temperature below 20° C. After 24h, additional 0.18 g (0.1 equiv, used total 1.6 equiv) of acid chloride12 was added and the reaction was continued to stir at room temperatureovernight (total 43 h). LC-MS analysis at 215 nm showed 43% of thedesired product (13) and ˜5% of compound 7. The reaction was dilutedwith dichloromethane (100 mL). The organic phase was washed withsaturated aqueous NH₄Cl (100 mL), water (100 mL) and saturated aqueousNaHCO₃ (100 mL). The organic phase was separated, dried over Na₂SO₄,filtered and concentrated to give crude product. The crude product waspurified by column chromatography eluting with 25-50% EtOAc in hexanesto afford compound 13 (1.63 g, 57% yield, HPLC area 93% pure) as a whitesolid. ¹H NMR in DMSO-d6: δ 11.19 and 10.93 (two singlets withintegration ratio of 1.73:1, total of 1H, same proton of two rotamers),7.93 (d, 2H), 7.66 (d, 2H), 5.80 (s, 2H), 3.36 (s, 2H), 3.27 (s, 2H),1.18 (s, 2H), 0.27 (q, 1H), 0.06 (s, 1H); Mass Spec: 435.0 (M+H)⁺

Step B. Synthesis of ST-246 (Route V)

Anhydrous DMF (6 mL) was added to a mixture of compound 13 (0.2 g, 0.46mmol), methyl 2, 2-difluoro-2-(fluorosulfonyl)acetate (0.44 mL, 3.45mmol, Aldrich) and copper (I) iodide (90 mg, 0.47 mmol). The reactionmixture was stirred at −90° C. for 4 h. LC-MS analysis at 254 nmindicated no starting material 13 remained and showed 48% HPLC area ofST-246. The reaction mixture was cooled to 45° C. and DMF was removedunder reduced pressure. The residue was slurried in EtOAc (30 mL) andinsoluble solid was removed by filtration. The filtrate was concentratedand purified by column chromatography eluting with 25-35% EtOAc inhexanes to afford ST-246 (55 mg, 32% yield, 95% pure by HPLC at 254 nm)as off-white solid. Analytical data (¹H NMR and LC-MS) were consistentwith those of ST-246 synthesized according to WO04112718.

What is claimed is:
 1. A method for producingN-[(3aR,4R,4aR,5aS,6S,6aS)-3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)-yl]-4-(trifluoromethyl)-benzamide,wherein said method comprises: (a) reacting compound 4 of formula:

with maleic anhydride to form compound 9 of formula:

(b) reacting compound 9 with cycloheptatriene; and (c) collectingN-[(3aR,4R,4aR,5aS,6S,6aS)-3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)-yl]-4-(trifluoromethyl)-benzamide.2. The method of claim 1, wherein step (a) is carried out in o-xyleneand reactants heated to reflux.
 3. The method of claim 1, wherein step(b) is carried out in toluene at a temperature of at least 75° C.
 4. Themethod of claim 1, wherein saidN-[(3aR,4R,4aR,5aS,6S,6aS)-3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)-yl]-4-(trifluoromethyl)-benzamidecollected in step (c) is further purified by column chromatography. 5.Compound 9 of formula: